1. Field of the Invention
This invention relates to a laser scanning microscope capable of scanning a laser beam onto a sample dyed with a fluorescence indicator and detecting fluorescence from the sample, and, an indicator discriminating method for discriminating the kind of fluorescence indicator dyeing the sample by using absorption wavelength spectral characteristics of the detected fluorescence.
2. Description of the Related Art
A laser scanning microscope is disclosed in, for example, Jpn. Pat. Appln. KOKAI Publication No. 2000-56244 and Jpn. Pat. Appln. KOKAI Publication No. 2002-55284. FIG. 9 shows a structure of the laser scanning microscope of Jpn. Pat. Appln. KOKAI Publication No. 2000-56244. The laser scanning microscope comprises a microscope 1 and a scanning device 2. The microscope 1 comprises a light source 3, an illumination lens system 4, a beam splitter 5, an objective 6, a stage 7 over which a sample S is placed, a condenser 8, a light source 9, a transmitted light detector 10, an image-forming lens 11, a beam splitter/mirror 12 and an eyepiece 13.
The scanning device 2 comprises a laser unit 14, a shutter 15, a collimation lens system 16, beam splitters 17 and 18, a scanning means 19, a confocal pinhole 20, a grating 21, image-forming mirrors 22 and 23, a one-dimensional change mirror array (DMD) 24, a converging lens system 25, and a detector 26.
The confocal pinhole 20 is provided at a conjugate position via the objective 6. A monochromator is composed of the grating 21 and the image-forming mirrors 22 and 23. The confocal pinhole 20 serves as an incidence opening of the monochromator. The change mirror array 24 is provided on a focal plane of the grating 21.
The laser unit 14 outputs a laser beam. The laser beam is passed through the shutter 15, the collimation lens system 16 and the beam splitters 17 and 18 and made incident on the scanning means 19. The laser beam is scanned, for example, in a direction X-Y by the scanning means 19. The laser beam scanned in the direction X-Y is reflected by the beam splitter/mirror 12, passed through the image-forming lens 11, the beam splitter 5 and the objective 6, and scanned over the sample S placed on the stage 7.
Fluorescence emitted from the sample S is made incident on the beam splitter/mirror 12 after passing through the objective 6, the beam splitter 5 and the image-forming lens 11. The fluorescence is reflected by the beam splitter/mirror 12, passed through the scanning means 19 and the beam splitter 18, and made to come into a focus on the confocal pinhole 20. The confocal pinhole 20 serves as an incidence opening of the monochromator composed of the grating 21, and the image-forming mirrors 22 and 23. The light beam from the sample S is split into spectral components by the dispersion effect of the monochromator.
The one-dimensional change mirror array 24 which can be arbitrarily programmed is, at least, provided on the focal plane of the grating 21. Thus, an image of the spectral light beam, from the sample S is optically formed on the change mirror array 24, converged by the converging lens system 25 and detected by the detector 26. By programing the change mirror array 24 in the above-described manner, the fluorescence wavelength spectral characteristics of the sample S can be obtained.
FIG. 10 shows a structure of the scanning microscope disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2002-55284. Laser beam L1 output from a laser light source 30 is spectrally spread by an optical device 31 to be wideband spectral illumination light L2. The spectral illumination light L2 is introduced into an AOTF (acousto-optic tunable filter) 32 and wavelength-selected. The wavelength-selected spectral light L3 is passed through a beam splitter 34, a scanning mirror 35 and an objective 36 and applied onto a sample S. The light beam reflected or emitted from the sample S returns in a direction reverse to the path for irradiation onto the sample S. Thus the light beam is passed through the beam splitter 34 and made incident on a detector 37.
As the optical device 31 outputs wideband spectral illumination light L2, the light quantity needs to be constant by selecting the wavelength. For this reason, an acousto-optically or electro-optically tunable filter (AOTF) may be combined with an acousto-optic or electro-optic deflector (AOD) and an acousto-optic or electro-optic beam splitter (AOBS) disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2001-124997. The acousto-optic or electro-optic deflector (AOD) and the acousto-optic or electro-optic beam splitter (AOBS) can be employed to select the wavelength and stop down the detected light.
In observation of the sample S dyed with the fluorescence indicator, the kind of the fluorescence indicator needs to be discriminated. In Jpn. Pat. Appln. KOKAI Publication No. 2000-56244, the fluorescence from the sample S is split into spectral contents by the dispersion effect of the monochromator composed of the grating 21 and the image-forming mirrors 22 and 23 and the light quantity in each of the wavelengths of the spectrum is detected by the detector 26. The quantity of the fluorescence from the sample S is small. The quantity of light in each of the wavelengths is further reduced by splitting the fluorescence from the sample S into the spectral contents. The SN ratio of detecting the fluorescence by the detector 26 is smaller. For this reason, the detected quantity of the fluorescence is very small and a fluorescent image becomes dark. If the quantity of the fluorescence is too small, the quantity of light in each of the wavelengths cannot be detected by the detector 26. As a result, the kind of fluorescence indicator dyeing the sample S cannot be discriminated with the fluorescence wavelength spectral characteristics.
Jpn. Pat. Appln. KOKAI Publication No. 2002-55284 does not suggest anything about discrimination of the kind of fluorescence indicator dyeing the sample S.